The human red blood cell has a uniquely developed membrane, which survives cellular deformations and shear stresses not encountered by most other cell types in the body. This membrane integrity and flexibility results from a two-dimensional protein network (membrane skeleton) on the cytoplasmic face of the membrane, which is comprised primarily of spectrin tetramers that crosslink short actin filaments. However, key structure-function features of spectrin tetramers and the spectrin self-assembly process are poorly understood. Equally important is the fact that the mechanisms by which hereditary hemolytic anemia mutations disrupt spectrin assembly and structure are in most cases inferred with little or no real supporting experimental data. The broad, long range goal of this project is to develop a detailed understanding of the mechanism of spectrin self-assembly into heterodimers and tetramers and determine how specific HE and HPP mutations perturb these critical molecular interactions, resulting in red cell membrane destabilization. Several hypotheses will be tested in these studies. The first hypothesis is that most HE mutations in ?-spectrin that are substantial distances from the tetramer site destabilize membranes by affecting the close?open dimer equilibrium. The second hypothesis is that tetramer assembly is substantially more complex than current three helix models suggest, with important contributions from Mg2+, terminal non-homologous ? and ? sequences, and possibly other co-factors including phosphorylation. The third hypothesis is that the weak lateral associations outside the dimer nucleation site are relatively non-specific, but they make critical contributions to the molecular properties of spectrin. The Specific Aims of the current proposal are: 1) determine how HE and HPP mutations affect tetramer assembly and destabilize red cell membranes; 2) define structural and functional roles of non-homologous spectrin tails and cofactors on spectrin tetramerization; 3) determine crystallographic structures of the spectrin tetramer site and interpret the effects of HE mutations on tetramer formation; and 4) determine inter-subunit docking and medium resolution structures of weak lateral ??? associations adjacent to the dimer initiation site. These Aims will be achieved using: thermodynamic analyses such as isothermal titration, sedimentation equilibrium and other biophysical methods to characterize intermolecular interactions; chemical crosslinking and mass spectrometry to define distance constraints that can distinguish between alternative molecular docking and conformational models, and X-ray crystallography to determine high resolution structures of non-covalent spectrin complexes. Structures of heterodimer and tetramer binding site complexes and elucidation of wild type interaction thermodynamics will provide a reference for determining how specific HE and HPP mutations disrupt spectrin self-assembly and thereby destabilize cell membranes. It is anticipated these studies will provide a solid foundation for genetic counseling as well as potential future development of novel therapeutic treatments for specific HE and HPP mutations.